Project Summary Iron-iron hydrogenases are highly active hydrogen-producing enzymes. Hydrogenases mediate molecular processes of broad biological and medical significance: the evolution and transport of gases and the transfer of electrons through FeS clusters. Hydrogenases lie at the heart of the metabolism of several anaerobic parasites, including the human parasite Trichomonas vaginalis. The coupling of hydrogenases to photosynthesis offers the possibility of the clean production of H{2} by engineered microorganisms. However, the connection of photosynthesis to hydrogenases is complicated by the sensitivity of the active sites of hydrogenases to oxygen. The specific aims of this proposal are: Aim 1: Directed Evolution of O{2}-tolerant Hydrogenases using Genetic Screens Aim 2: Construction, Characterization and Evolution of a Library of Novel FeFe-hydrogenases Aim 3: Characterization and Evolution of Hydrogenase Accessory Proteins